The challenge to create the microenvironment enabling growth of an in-vitro microtissue perfused with living microvessels (e.g., arterioles, capillaries, and venules) represents a completely new paradigm in the creation of 3-D tissues. By definition, a 3-D tissue requires enhanced transport of nutrients and waste relative to 2-D monolayer cultures. Current approaches to create such an environment have employed three primary approaches: 1) enhanced concentration gradients of nutrients and waste while relying on molecular diffusion (Brownian motion) as the mode of transport, 2) the creation of microchannels in the tissue to enhance advection (forced convection), or 3) forced interstitial fluid flow. In-vivo, diffusion of nutrients and waste is the mechanism of transport once solutes exit the capillary bed, and is generally limited to distances <250 μm. The rate of transport is proportional to the concentration difference between two points, and inversely related to the separation distance. Hence, numerous 3-D tissue models have been reported with dimensions on the order of 1-10 mm by simply enhancing the oxygen tension (room air is 160 mmHg compared to 20-30 mmHg in the interstitial tissue) and concentration of other nutrients (e.g., glucose).
More recently, microfabrication technology has led to the creation of precise microchannels on non-biological substrates (e.g., silicon or polydimethyl siloxane, PDMS)1, 2, or within biological substrates such as collagen3. While these approaches offer the distinct advantage of introducing advection as a mechanism of transport, even when “endothelialized”, the channels are not living microvessels. Hence, while this approach may assist the creation of larger engineered tissues, they are of less benefit in understanding in vivo biological functions such as angiogenesis, cell migration, cell differentiation, and ischemia.
Interstitial fluid flow can markedly impact extracellular gradients of solutes, enhance transport of nutrients and waste, and significantly impact the development of both lymphatic and blood capillaries4-6. These recent studies, as well as others including our group7-9, highlight the ability to generate living microvessels in 3-D, and also demonstrate that these living microvessels can become functional upon implantation. However, perfusion of human (or other animal) microvessels in-vitro has not been demonstrated.
In short, there have been no reports describing successful creation of actual living microvessels that are perfused with an appropriate fluid to deliver nutrients to a 3-D tissue.